A novel oncolytic adenovirus targeting to telomerase activity in tumor cells with potent

Abstract

Telomerase is a therapeutic target for cancer. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionaly upregulated exclusively in about 90% of cancer cells. Previous studies have demonstrated that hTERT promoter can control the expression of exogenous genes to the telomerase-positive cancer cells, thus hTERT promoter is an excellent candidate for generating cancer-specific oncolytic adenovirus. In this study, we devised a novel oncolytic adenovirus (Ad.TERT) by replacing the normal E1A regulatory elements with hTERT promoter. Ad.TERT displays cancer-specific E1A expression, virus replication and cytolysis in in vitro experiments. In animal experiments, intratumoral administration of Ad.TERT demonstrates potent antitumoral efficacy at least in two xenograft models (Bcap37 and BEL7404). Ad.TERT was targeted by the telomerase activity in cancer cells and has potent antitumoral efficacy in vivo, and since telomerase activity is a wide-ranged tumor marker, Ad.TERT could be a powerful therapeutic agent for a variety of cancers.

Introduction

Oncolytic adenoviruses are a class of promising anticancer agents (Alemany et al., 2000; Curiel, 2000; Kirn et al., 2001; Kruyt and Curiel, 2002), which replicate selectively in cancer cells, resulting in cancer-specific cytotoxicity. One strategy to construct oncolytic adenoviruses is to use tumor or tissue-specific promoters to control the expression of viral genes essential for replication, resulting in the expression of viral genes selectively in cancer cells, thus, the virus should only replicate in and kill these cells (Rodriguez et al., 1997; Hallenbeck et al., 1999; Kurihara et al., 2000; Matsubara et al., 2001; Li et al., 2001; Yu et al., 2001; Jakubczak et al., 2003; Post and Van Meir, 2003).

Adenoviral E1A gene products stimulate S-phase entry and transactivate both viral and cellular genes that are critical for viral replication (Whyte et al., 1988). Given the essentiality of E1A, controlling the expression of E1A gene is a good approach for restricting the replication of adenovirus to tumor cells. The choice of promoters is important for constructing oncolytic adenovirus. The used vectors replicated selectively in some types of cancer cells and replication-attenuated in normal cells; however, their applications are restricted to only a narrow range of tumors as these promoters are only active in the type of cancer from which they are derived (Nettelbeck et al., 2000). To overcome this problem, promoters operative widely in a variety of tumors are desired.

Recently, telomerase has been recognized as a new wide-range tumor marker (Blackburn, 1991; Komata et al., 2002). It has been reported that telomerase activity is significantly higher in about 90% of cancers and correlates well with the degree of malignancy (Kim et al., 1994; Shay, 1997). Human telomerase has three subunits (Feng, 1995; Meyerson et al., 1997; Nakayama et al., 1997), among them, the catalytic subunit of telomerase, hTERT, is the determinant of the telomerase activity. Moreover, the expression of hTERT is transcriptionally upregulated in a variety of cancer cells while repressed in normal cells (Cong et al., 1999; Takakura et al., 1999). Therefore, its promoter region has been cloned and used in targeting cancer gene therapy to restrict the expression of exogenous genes to cancer cells (Gu et al., 2000,2002; Plumb et al., 2001; Lin et al., 2002; Bilsland et al., 2003), including our previous work (Liu et al., 2002).

We speculated that hTERT promoter could be an excellent candidate for generating cancer-specific oncolytic adenovirus. In this study, we described a novel oncolytic adenovirus Ad.TERT, in which E1A gene is under the control of hTERT promoter, and demonstrated that Ad.TERT specifically replicated in and killed a panel of tumor cells, but not normal cells. Moreover, direct injection of Ad-TERT into two tumor xenograft models resulted in potent antitumoral efficacy. Ad.TERT offers a promising treatment platform for a variety of cancers of which the hallmark is high telomerase activity.

Materials and methods

Cells and culture

Human breast cancer cell line Bcap37, a highly malignant and heat-resistant human breast cancer cell line, purchased from Shanghai Cell Collection, Chinese Academy of Sciences (CAS) (Wang et al., 2002). Human hepatocarcinoma cells Hep 3B and BEL7404, human cervical caner cell line Hela, human colorectal cancer cell line SW620, human lung cancer cell line A549, human normal embryonic lung cells WI38, human lung fibroblasts NHLF, and human normal embryonic lung cells MRC5 were purchased from American Tissue Culture Collection (ATCC, Rockville, MD, USA). The HEK293 cell line (human embryonic kidney containing the E1A region of Ad5) was obtained from Microbix, Inc (Ontario, Canada). MRC5, Hela, Hep3B, BEL7404, SW620 and HEK293 cells were cultured in DMEM (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO BRL), 4 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. Bcap37, WI38, and NHLF cells were grown in RPMI1640 supplemented with 10% FBS.

Luciferase assay

The hTERT promoter (−378 bp/+78 bp) was obtained from pAd/hTERT, a gift from Dr Bingliang Fang (University of Texas MD Anderson Cancer Center, Houston, TX, USA). The luciferase reporter plasmid pGL3-hTERT was constructed by inserting hTERT promoter into the pGL3-Basic. The cells were plated at a density of 1.0 × 105 cells/ml and transfected with pGL3-hTERT using LipofectAMINE (GIBCO-BRL) 24 h later. A promoter-free luciferase plasmid (pGL3-Basic) and a plasmid containing the expression cassette SV40 enhancer/promoter-luciferase (pGL3-control) were also transfected under the same condition. At 48 h post-transfection, cells were washed twice with phosphate-buffered saline (PBS) and lysed in the report lyses buffer (Promega, Madison, WI, USA). The luciferase activity was assayed by the dual-luciferase reporter assay system (Promega Madison, WI, USA).

Virus construction

pXC1 (Microbix Biosystems, Ontario, Canada) contains the Ad5 bp 22–5790, including the inverted terminal repeat, the packaging sequences, and the E1A and E1B genes inserted to pBR322 (McKinnon et al., 1982). The regions of E1A promoter (342–552) was deleted by polymerase chain reaction (PCR) using pXC1 as the template. The viral region comprising nucleotides 22–342 was amplified by PCR using the following primers: 5′IndexTermtcctgtggatccgggcccccatttc3′ and 5′IndexTermttcagtacgtagtcgacctcgagatattacgcgctatgagtaacac3′. In a similar way, the region containing nucleotides 552–1359 was amplified with the following primers: 5′IndexTermtactactattgcattctctagacaca3′ and 5′IndexTermtatctcgaggtcgactacgtactgaaaatgagacatattatc3′. Both PCR products contain a 23-bp random sequence at the 5′ end, which is complementary to each other. This random sequence comprises restriction enzyme sites of I, SalI, and SnaBI. Using the mixture of these two PCR fragments as template, a fragment of about 1100 bp was amplified using the primers 5′IndexTermtcctgtggatccgggcccccatttc3′ and 5′IndexTermtactactattgcattctctagacaca3′. The PCR product was digested with BamHI/XbaI and ligated into BamHI/XbaI sites of pXC1 to construct the plasmid pZXC2.

hTERT promoter was amplified from pAd.hTERT using the following primers: 5′IndexTermtcttctcgagtggcccctccctcgggttac3′ and 5′IndexTermgctacgtacgcgggggtggccggggcca3′. The PCR product of about 460 bp was digested with XhoI/SnaBI, and then cloned into the XhoI/SnaBI sites of pZXC2, resulting in the plasmid pZhTERT. In summary, pZhTERT contains the Ad5 bp 22–5790, with E1A regulatory elements (bp 342–552 region) replaced with hTERT promoter. The sequence of the fragment was confirmed by DNA sequencing.

Oncolytic adenovirus (Ad.TERT) was constructed by standard homologous recombination techniques using the plasmid pZhTERT and the adenovirus packaging plasmid pBHGE3 (adenovirus packaging plasmid, Microbix Biosystems, Ontario, Canada) in HEK293 cells. pZhTERT (1 μg) was mixed with 0.5 μg pBHGE3 and then cotransfected into HEK293 cells using Effectene Transfection Reagent (Qiagen, Germany). Recombinant adenovirus was isolated from a single plaque and expanded in HEK293 cells.

Control adenovirus Ad.WT (wild-type Ad5) is constructed by standard homologous recombination techniques using the plasmid pXC1 and the adenovirus packaging plasmid pBHGE3. Ad.EGFP (a replication-defective Ad5 devoid of E1 and E3 but containing the cytomegalovirus immediately early promoter driving the EGFP gene) and Ad.β-gal (a replication-defective Ad5 devoid of E1 and E3 but containing the cytomegalovirus immediately early promoter driving the β-galactosidase gene) were kindly provided by Dr Qijun Qian (Second Military Medical University, Shanghai, China). Large-scale preparations of adenovirus were purified by centrifugation banding on cesium chloride, and the titers were determined by plaque assay on HEK293 cells.

PCR verification of the virus

To verify the sequences of Ad5WT and Ad.TERT, DNA was extracted from purified viruses with QIAamp DNA Blood Mini Kit (Qiagen, Germany) and analysed by PCR. The used primers were specific for the adenoviral ‘left’ inverted terminal repeat (ITR) (Ad5 nucleotides 277–297, 5′IndexTermcgcgggaaaactgaataagag3′, primer 1) and E1A open reading frame (ORF) (Ad5 nucleotides 759–779, 5′IndexTermaccgccaacattacagagtcg3′, primer 2). The resulting fragments were resolved in a 1.5% agarose gel and visualized with an UV transilluminator.

Western blot analysis of E1A expression

Cells were infected with Ad.TERT, Ad.EGFP, and Ad5.WT at MOI of 10. At 48 h postinfection, cells were washed with PBS, and then lysed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10 glycerol, 1.55% DTT). Total cell lysates (30 μg of protein) were separated by 12% SDS–PAGE and then transferred to nitrocellulose membranes (Amersham). The levels of E1A protein was determined by Western blot analysis with the rabbit polyclonal antibody anti-Ad2 E1A (SC-403, Santa Cruz Biotech). The reactivity was visualized by enhanced chemiluminescence (ECL) system (Amersham life Sciences Inc., Arlington Heights, IL, USA).

Cytotoxicity assay

For Figure 4a, cells were seeded at 40 000/well in 24-well plates, and 24 h later infected with Ad.TERT, Ad.EGFP, and Ad5WT at various MOIs. After 7 days, the appearance of cytopathic effect (rounding and detachment) was monitored and documented as photographs. For Figure 4b, tumor cells and normal cells were uninfected or infected with Ad.TERT and Ad.WT at various MOIs. After 9 days, cells were exposed to 2% crystal violet in 20% methanol for 15 min, then washed with distilled water and documented as photographs.

Figure 4
figure4

Tumor-selective cytotoxicity of Ad.TERT. (a) Subconfluent tumor cells (Bcap37, BEL7404, Hela, and Hep3B) and confluent normal cells (NHLF and MRC5) were infected with Ad.TERT, Ad.EGFP, and Ad.WT. MOIs used are shown in parentheses. Mock-infected cells were included as a control. After 7 days, the appearance of cytopathic effect (rounding and detachement) was monitored, and documented as photographs. (b) Subconfluent BEL7404 and confluent NHLF cells were not infected or infected with Ad.TERT and Ad.WT at the indicated MOIs. After 9 days, cells were stained with crystal violet, and documented as photographs

Virus progeny production assay

To determine virus progeny production, 3 × 105 cells were seeded in six-well plates. After 24 h, cells were infected with 104 PFU of Ad.TERT and Ad.WT. After an additional 48 h, medium and cells were scraped into 1.5 ml medium, after three thaw cycles, centrifuged to collect the supernatant. The production was determined by standard plaque assay on 293 cells.

X-gal staining of cells in culture

To analyse the complementation of the E1a deficiency of adenoviral vectors with Ad.TERT, cells were seeded at 24-well plates, and 24 h later coinfected with an E1E3 vector expressing β-galactosidase (Ad.β-gal, MOI off 2) and Ad.TERT, Ad.EGFP, or Ad5WT (MOI of 1). After 5 days, the cells were stained with 5-bromo-4 chloro-3-indolyl-β-D-galactopyranoside (X-gal), and photographed.

Animal experiments

Female Balb/c nude mice at 4–5 weeks were obtained from the Animal Research Committee of the Institute of Biochemistry and Cell Biology (Shanghai, China) and quarantined for a week before tumor implantation. For Figure 6a, the xenografts tumor model were established by subcutaneously injecting 1 × 106 BEL7404 cells into the right flank of mice. On day 20, when the tumors reached about 150 mm3, animals were randomly grouped into PBS (n=7), Ad.EGFP (n=6), Ad.WT (n=7), and Ad.TERT (n=7) groups. A daily dose of 5 × 108 PFU virus in 100 μl of PBS was administrated intratumorally every other day for three times. The tumor size was monitored for the following 45 days. The tumor volumes were estimated as tumor volume (mm3)=length × width2/2. For Figure 6b, the xenografts tumor model were established by subcutaneously injecting 1 × 106 Bcap37 cells into the right flank of mice. On day 10, when the tumors reached 50–100 mm3, animals were randomly grouped into PBS (n=5), Ad.WT (n=5), and Ad.TERT (n=6) groups. A daily dose of 5 × 108 PFU in 100 μl of PBS was administrated intratumorally every other day for three times. The tumor size was monitored for the following 45 days.

Figure 6
figure6

Ad.TERT has potent antitumoral efficacy on BEL7404 and Bcap37 xenograft tumors. (a) Tumor xenografts were established by injecting 1 × 106 BEL7404 cells suspended in 100 μl of serum-free DMEM into the right flank of nude mice. On day 20, when tumor volumes reached about 150 mm3, mice were randomized and intratumoraly injected with 5 × 108 PFU of Ad.EGFP (n=6), Ad.TERT (n=7), Ad.WT (n=7), and PBS diluents (n=7) for three times every other day. (b) Tumor xenografts were established by injecting 1 × 106 Bcap37 cells suspended in 100 μl of serum-free DMEM into the right flank of nude mice. On day 10, subcutaneous tumors of about 50–100 mm3 were intratumorally injected with 5 × 108 Ad.TERT (n=6), Ad.WT (n=5), or with PBS (n=5) for three times every other day. The tumor volumes were estimated as tumor volume (mm3)=length × width2/2

Results

hTERT promoter is active in tumor cells

To compare the telomerase activity in tumor and normal cells, TRAP-PCR-ELISA was performed in six tumor cell lines (Bcap37, BEL7404, Hep 3B, A549, Hela, and SW620) of various origins (breast cancer, hepatocarcinoma, lung cancer, and colorectal cancer) and three normal cell lines (NHLF, MRC-5 and WI38). It was verified that all the tumor cell lines showed significantly higher telomerase activity despite some variation, while three normal cell lines did not show detectable telomerase activity (data not shown).

Using the luciferase reporter assay, we assessed hTERT promoter activity in tumor and normal cells. As shown in Figure 1, hTERT promoter showed significant transcriptional activity in all the telomerase-positive tumor cells (SW620, Bcap37, BEL7404, Hep3B, A549, and Hela), whereas its transcriptional activity was much lower in the telomerase-negative normal cells (NHLF, WI38, and MRC5). These results proved that the hTERT promoter could drive the expression of exogenous genes specifically in tumor cells.

Figure 1
figure1

Transcriptional activity of the hTERT promoter in tumor or normal cells. The hTERT activity in each cell line was calculated as percentage of the positive control (pGL3-control). Then the hTERT promoter activity in SW620 cells was considered as 100, transcriptional activity in other cell lines was calculated against that in SW620 cells. pGL3-basic without enhancer/promoter was used as a negative control. The data shown are MEAN±s.d. (bars) values (n=3)

Construction of Ad.TERT virus

hTERT promoter is active in tumor cells from various origins, but silent in normal cells (Figure 1). This makes hTERT promoter a promising candidate for constructing tumor-specific oncolytic adenoviruses. For this purpose, we constructed Ad.TERT, a cancer-targeted, replication-competent adenovirus, by replacing the normal E1A regulatory elements with hTERT promoter. The E1A promoter was deleted by PCR strategy, and replaced with the unique restriction enzyme sites, where hTERT promoter was introduced. Figure 2 depicts the genome of the recombinant adenoviruses. In Ad.TERT, E3 region was reserved, which was reported to enhance the viral spread and oncolysis (Yu et al., 1999). The viral DNA was extracted and the modified sequences were confirmed by PCR (data not shown).

Figure 2
figure2

Schematic structure of Ad.TERT. In Ad.TERT, the normal E1A promoter (bp 342–552) was replaced by hTERT promoter (−378 bp/+78 bp). The dark boxes represents the inverted terminal repeats (ITR) and Ψ the adenovirus 5 packaging signal. Primers 1 and 2 were used to analyse the sequences of Ad.WT and Ad.TERT

Ad.TERT mediates tumor-specific E1A expression

To ensure the specificity of hTERT promoter in the context of oncolytic adenoviruses, a panel of human tumor cells and normal cells were infected with Ad.TERT at an MOI of 10. After 48 h incubation, the expression of E1A protein was assessed by Western blot assay. The results in Figure 3 demonstrated that the expression of E1A could be detected in Ad.TERT-infected tumor cells (Bcap37, BEL7404, Hep 3B and Hela), but not in normal cells (NHLF and MRC5) (lane 2). As the controls, the cells were also infected with Ad.EGFP (a replication-defective Ad5 virus expressing EGFP under the control of the CMV promoter) or with Ad.WT (wild-type Ad5). There was no detectable E1A expression in cells uninfected or infected with Ad.EGFP (lanes 1 and 3), while Ad.WT infection caused the significant expression of E1A in all cell lines (lane 4). These findings confirmed that Ad.TERT mediates selective expression of E1A in tumor cells, but not in normal cells.

Figure 3
figure3

Western blot assay of E1A expression by Ad.TERT. Cells were infected with Ad.TERT (lane 2), Ad.EGFP (replication-defective Ad5 expressing EGFP gene, lane 3), Ad.WT (wild-type Ad5, lane 4); mock-infected cells were included as a control (lane 1). Lysates from control and infected cells were subjected to western blot assay with anti-Ad2 E1A Ab as described in Materials and methods

Ad.TERT induces tumor-specific cytotoxicity

Encouraged by the specificity of E1A expression mediated by Ad.TERT, we sought to evaluate the cytotoxity of the Ad.TERT. A set of tumor cell lines (Bcap37, BEL7404, Hep 3B, and Hela) and normal cell lines (NHLF and MRC5) were infected with Ad.TERT, Ad.EGFP, and Ad.WT at various MOIs. After 7 days, cells were examined regularly for CPE. Figure 4a depicts representative assay results. As expected, Ad.EGFP caused no cytotoxicity in all cells, while Ad.WT induced cell killing in both tumor cells and normal cells with no specificity. In contrast, Ad.TERT infection had selective cytotoxicity on tumor cell lines, but had no significant CPE on any normal cells. The morphology of Ad.TERT-infected normal cells appeared to be similar with uninfected cells.

To further monitor the cytotoxicity of Ad.TERT, tumor (BEL7404) and normal (NHFL) cells were uninfected or infected with Ad.TERT and Ad.WT at various MOIs. After 9 days, the cytotoxicity was detected by staining adherent cells with crystal violet. As shown in Figure 4b, at all titers the cytotoxicity of Ad.TERT was nearly similar to Ad.WT in BEL7404 cells. However, Ad.TERT induced about 100-fold attenuation in killing NHFL cells relative to Ad.WT. Similar results were obtained from other tumor and normal cell lines (data not shown). Taken together, Ad.TERT showed nearly similar effect with Ad.WT in killing tumor cells. In contrast, the cytotoxicity of Ad.TERT to normal cells is much lower than Ad.WT.

Ad.TERT replicates selectively in tumor cells

According to the mechanism of oncolytic virus, the most important character of oncolytic virus is its ability of selectively replicating in tumor cells (Alemany et al., 2000). To compare the replication ability between Ad.TERT and that of wild-type adenovirus, tumor cells (Hep 3B, Bcap37, BEL7404, and Hela) and normal cells (NHLF and MRC5) were infected by 104 PFU of Ad.TERT and Ad.WT, and the cells lysates were tittered on 293 cells 48 h postinfection. In Hep 3B, Bcap37, BEL7404, and Hela cells, Ad.TERT replicated at levels comparable with Ad.WT. However, Ad.TERTs replication was severely attenuated in NHLF and MRC5 cells, producing 600- and 1000-fold less virus in NHLF cells and in MRC5 cells than Ad.WT, respectively (Figure 5).

Figure 5
figure5

Tumor-selective replication of Ad.TERT. (a) 3 × 105 cells were plated into six-well plates. After 24 h, the cells were infected with 104 PFU of Ad.TERT and Ad.WT. After an additional 48 h, medium and cells were scraped into 1.5 ml medium and subjected to three-thaw cycles. The collected supernatant was tested for virus production by standard plaque assay on 293 cells. Progeny viruses from 1 PFU of virus were calculated. The results were the average of two independent experiments. (b) Cells were seeded at 24-well plates and 24 h later coinfected with an Ad.β-gal (MOI of 2) and Ad.EGFP, Ad.TERT,or Ad.WT (MOI of 1). After 5 days, the cells were stained with X-gal and photographed

As another means to demonstrate the cancer-specific replication of Ad.TERT, we asked whether it could complement the replication of an E1E3 vector expressing β-galactosidase (Ad.β-gal) in cancer cells. Ad.β-gal could not replicate in cells; however, if a cell is coinfected with Ad.β-gal and a replication-competent adenovirus, the same regulation of replication can apply to both viruses because replication-competent adenovirus will provide E1A protein in trans. BEL7404 and MRC5 cells were infected with Ad.β-gal (MOI of 2) alone, or with Ad.β-gal plus Ad.EGFP, Ad.TERT, or Ad.WT (MOI of 1). After 5 days, the cells were stained with X-gal. With Ad.β-gal alone, or with Ad.β-gal plus Ad.GFP, only individual blue cells were obtained. With Ad.β-gal plus Ad.WT, many cells within the focus were stained with X-gal in both BEL7404 cells and MRC5 cells. Although the stained MRC5 cells is fewer than BEL7404 cells, the stained cellular area and intensity increased significantly in MRC5 cells than Ad.β-gal alone. With Ad.β-gal plus Ad.TERT, most of the cells were stained with X-gal in BEL7404 cells. However, only individual blue cells were stained in MRC5 cells. In summary, Ad.TERT complement the replication of Ad.β-gal in cancer (BEL7404) cells, but not in normal (MRC5) cells. This result is consistent with virus production assay. Similar results were obtained from other tumor (Bcap37) and normal (NHFL) cell lines (data not shown). These results confirmed the cancer-specific replication of Ad.TERT, and its replication properties correlate remarkably with its cytotoxicity.

Antitumoral efficacy of Ad.TERT in nude mice

hTERT promrter is active in more than 90% cancer cells and in vitro experiments showed that Ad.TERT could kill cancer cells from various origins. So we evaluated the antitumoral efficacy of Ad.TERT in different tumor models (BEL7404 and Bcap37 cells were used as examples in this paper).

Subcutaneous tumors were established in the right flank of nude mice using hepatocarcinoma (BEL7404) cells. Macroscopic tumor with a volume of approximately 150 mm3 were injected intratumorally with PBS, Ad.EGFP, Ad.TERT, and Ad.WT every other day for a total of three injections. As shown in Figure 6a, Ad.TERT significantly inhibited the tumor growth, and the antitumoral effect is comparable with Ad.WT. In contrast, replication-defective adenovirus (Ad.EGFP) has no effect in the tumor growth versus PBS—treated groups. This result is consistent with the cytotoxicity data and virus production data.

Ad.TERT also reduced the growth of human Bcap37 breast cancer in nude mice, which is a very aggressive xenograft (Wang et al., 2002). Tumors of about 50–100 mm3 were injected with PBS, Ad.TERT, and Ad.WT every other day for a total of three injections. As shown in Figure 6b, the tumor growth in the Ad.TERT-treated or Ad.WT-treated group was reduced versus the PBS-treated group. However, there was also a rapid increase in average tumor volume during the first 4 weeks after virus injection, reaching approximately 1000 mm3 on day 35. Interestingly, in the following days, the tumor volume rapidly decreased (Figure 6b), in some cases, the tumor bodies were completely elimilated (data not shown).

In vitro experiments showed that Ad.TERT has a similar effect with Ad.WT in killing tumor cells, and the cytotoxicity of Ad.TERT to normal cells is much lower than Ad.WT (Figure 4a and b). However, in animal experiments, Ad.TERT did not show improved therapeutic benefit compared with wild-type adenovirus (Figure 6a and b). In fact, the cytotoxicity of wild-type adenovirus to normal cells could not be shown in this animal experiments, because human adenovirus cannot replicate in mice cells. The limitations of animal model make it impossible to compare the safety between Ad.TERT and Ad.WT. So it is not strange that wild-type adenovirus has the same antitumoral effect with Ad.TERT. The limitation of animal model is a broad problem faced by oncolytic adenovirus research (Alemany et al., 2000; Curiel, 2000).

Discussion

In our study, we described a novel oncolytic adenovirus named Ad. TERT, in which hTERT promoter was used to restrict the E1A expression. Given that the telomerase activity is elevated in the vast majority of human tumors, Ad.TERT could be a powerful anticancer agent to a wide rage of human malignancies from different etiology.

According to the published data, hTERT promoter was once applied to control the E4 region of adenovirus (AdEHT2, Hernandez-Alcoceb et al., 2002), but AdEHT2 could not attenuate the replication of adenovirus in the telomerase-negative IMR-90 cells when infected under hypoxic conditions. It is interesting to compare the difference between our modified virus Ad.TERT and Ad.EHT2. Firstly, hTERT promoter was used to control the expression of E4 gene in Ad.EHT2. It is known that hTERT promoter can be potentially stimulated by adenoviral E1A protein, either directly or as a consequence of the activation of the cell cycle (Kirch et al., 2002). AdEHT2 virus could express E1A protein in IMR-90 cells, so the selectivity of hTERT promoter in IMR-90 cells may be lost owing to the E1A expression. In fact, the low expression of E4 mRNA can be noticed in Ad.EHT2-infected IMR-90 cells at 14 h after infection, and the level of expression was comparable with wild-type adenovirus-infected cells (Hernandez-Alcoceb et al., 2002). However, in our strategy, hTERT promoter was used to control the E1A expression. Ad.TERT did not express E1A proteins in normal cells but expressed selectively in tumor cells (Figure 3), indicating that the selectivity of hTERT promoter was reserved in Ad.TERT. We conclude that the selective expression of E1A results in selective viral replication, progeny production, and cytotoxicity. Similar results were reported from Ad.E2FRC (Tsukuda et al., 2002), in which the E1A expression is under the control of E2F-1 promoter, which is also known as being activated by E1A protein. Secondly, although the E4 region is believed to be necessary for virus replication, low levels of expression of the E4 ORFs may be supported by the replication of adenovirus. Thirdly, the length of hTERT promoter is different between AdEHT2 (bp-218 to +51) and Ad.TERT (–378 to +78). The used promoter in Ad.TERT is longer than that in AdEHT2. It is possible that the longer one has certain key silencer, which is missing in the shorter.

The major concerns about the hTERT promoter-driven oncolytic adenovirus ares their putative toxicity when Ad.TERT is delivered systemically, especially to some normal cells, in which the telomerase is also active, including some progenitor cells of bone marrow cells, hematopoetic, epithelial, and gut original cells, as well as hair follicles undergoing mitosis (Chiu et al., 1996; Yasumoto et al., 1996; Ramirez et al., 1997; Nakamura et al., 1999). Fortunately, these cells are poorly transfected by adenovirus according to the published work (Watanabe et al., 1996; Gu et al., 2000). The ineffectiveness of the adenovirus in transducing these cells supports the safety of Ad.TERT in systemic delivery. Another support for the less toxicity in vivo is that telomerase activity in those cells is only transient (Bodnar et al., 1996; Chiu et al., 1996), suggesting that toxic effects arising from Ad.TERT, if any, may be transient. Nevertheless, it is important to investigate the long-term toxic effects of Ad.TERT in systemic delivery. However, we could not do it in the nude mice, because mice are well known to be nonpermissive for human Ad. In the safety consideration of Ad.TERT, another fact should be noted that subgroup C Ad in nature is benign in adults except in case of severe immunodeficiency (Horwitz, 1996).

The major problem of gene therapy is the low transduction efficiency in vivo. One possibility to address this problem is the use of oncolytic virus as gene therapy vectors. Oncolytic virus could replicate in vivo and spread throughout the tumor tissue, thereby could deliver a high dose of a therapeutic gene product efficiently and selectively to tumor cells. Experimentally, gene delivery by an oncolytic virus reaches a larger area of tumor compared with that reached by replication-defective viral vector (Ichikawa and Chiocca, 2001). Combining a replicating adenovirus that expresses thymidine kinase with ganciclovir treatment enhanced anticancer activity (Freytag et al., 1998; Wildner et al., 1999). Additionally, cytokines cDNAs have also been added to the oncolytic adenovirus genome (Motoi et al., 2000). In our study, we incorporated reporter gene (EGFP) into the Ad.TERT genome. Experiments showed that Ad.TERT significantly increase the EGFP expression in BEL7404 cells than the replication-defective adenovirus, but not in MRC5 cells (data not shown). It is conceivable that Ad.TERT could be used to increase the efficiency of gene delivery.

In conclusion, we have proved that hTERT promoter could restrict the adenovirus replication to the telomerase-active tumor cells. hTERT promoter is active in cancers from very different etiology, indicating that Ad.TERT could be used in a wide range of human malignancies. Our preliminary research on constructing a highly specific and wide-ranged oncolytic adenovirus pave the way for our future studies.

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Acknowledgements

We thank Bingliang Fang for providing us with the hTERT promoter. We also thank Lanyin Sun for help in the cell culture, Weijing Xu and Binghua Li for critical reading and discussion of this paper. This work was supported by the Key Project of the Chinese Academy of Sciences (No. KSCX2-3-06), the National Natural Science Foundation of China (No.30120160823), and the State 863 High Technology R&D Project of China (No. 2001AA217031).

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Correspondence to Xinyuan Liu.

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Zou, W., Luo, C., Zhang, Z. et al. A novel oncolytic adenovirus targeting to telomerase activity in tumor cells with potent. Oncogene 23, 457–464 (2004). https://doi.org/10.1038/sj.onc.1207033

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Keywords

  • oncolytic adenovirus
  • E1A gene
  • hTERT promoter
  • telomerase
  • cancer

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